DPH1 and DPH2 variants that confer susceptibility to diphthamide deficiency syndrome in human cells and yeast models

ABSTRACT The autosomal-recessive diphthamide deficiency syndrome presents as intellectual disability with developmental abnormalities, seizures, craniofacial and additional morphological phenotypes. It is caused by reduced activity of proteins that synthesize diphthamide on human translation elongation factor 2. Diphthamide synthesis requires seven proteins (DPH1-DPH7), with clinical deficiency described for DPH1, DPH2 and DPH5. A limited set of variant alleles from syndromic patients has been functionally analyzed, but databases (gnomAD) list additional so far uncharacterized variants in human DPH1 and DPH2. Because DPH enzymes are conserved among eukaryotes, their functionality can be assessed in yeast and mammalian cells. Our experimental assessment of known and uncharacterized DPH1 and DPH2 missense alleles showed that six variants are tolerated despite inter-species conservation. Ten additional human DPH1 (G113R, A114T, H132P, H132R, S136R, C137F, L138P, Y152C, S221P, H240R) and two DPH2 (H105P, C341Y) variants showed reduced functionality and hence are deficiency-susceptibility alleles. Some variants locate close to the active enzyme center and may affect catalysis, while others may impact on enzyme activation. In sum, our study has identified functionally compromised alleles of DPH1 and DPH2 genes that likely cause diphthamide deficiency syndrome.

(A) expression in yeast: Total protein extracts of yeast strains expressing Dph1 or DPH2 variants were subjected to Western blot analyses with a polyclonal antibody that detects human and yeast Dph1 (ABIN2784704, Antibodies-Online).Protein extracts of the wild-type and dph1∆ served as controls, and yeast pyruvate kinase Cdc19 as independent loading control.lower panel: Yeast strains genomically expressing Dph2 variants harboring Cterminal (c-myc)3 tags were applied to Western blot detection of Dph2 with anti-c-myc antibodies (9E10) according to (Janke et al., 2004).Protein extracts of the wild-type expressing Dph2 with no tag and a strain expressing wild-type Dph2-c-myc, and detection of the yeast pyruvate kinase Cdc19 served as controls.

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B) expression in MCF7: Expression of plasmid-encoded DPH1 cDNA in DPH1ko MCF7 cells and DPH2 cDNA in DPH2ko MCF7 cells.Total protein extracts of DPH1ko MCF7 cells transiently transfected with cDNA encoding DPH1 or -variants were subjected to Western blot analyses with a polyclonal antibody (Sigma-Aldrich cat.no.F7425) that detects the Flag tag added to the C-termini of DPH1.Total protein extracts of DPH2ko MCF7 cells transiently transfected with cDNA encoding DPH2 or -variants were subjected to Western blot analyses with a polyclonal antibody (Sigma-Aldrich cat.no.F7425) that detects the Flag tag added to the C-termini of DPH2.Disease Models & Mechanisms: doi:10.1242/dmm.050207:Supplementary information Disease Models & Mechanisms • Supplementary information

Fig. S3 .
Fig. S3.Sensitivity of yeast strains that express wildtype or variant DPH1 or DPH2 upon exposure to Diphtheria toxin.Phenotypic spot assay of DPH1 and DPH2 strains under exposure towards Diphtheria toxin (DT).A), B) and C): Strains were transformed with a galactose-inducible construct expressing the catalytic subunit of DT (pSU9 according to Uthman et al, (2013)).Ten-fold serial dilutions were spotted on minimal Yeast Nitrogen Base media lacking uracil with either 2% glucose or 2% galactose as carbon sources and incubated at 30°C for three days to visualize survivors in the presence of DT.

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Fig. S4.MCF7-based assays apply ADPR instead of noDiphth antibody to assess enzyme activity because transiently transfected diphthamide deficient MCF7 cell preparations still contain a high background of non-diphthamidylated eEF2.

Table S1 . Mutations of DPH1 and DPH2 in diphthamide deficiency syndrome patients.
HumanDPH1 mRNA carries 2 translation start codons, the first preceding the 2 nd by 5 amino acids (MrrqvMaalvv…), the listed variant positions refer to translation from the 1st (see methods).Activities were assigned according to ADPR assays in MCF7 and in yeast with western blot (WB) and diphtheria toxin sensitivity (DT) assays, with 'active' being indistinguishable from the WT control or (*) with just minimal signals upon long exposures, 'reduced' with detectable decrease but relevant remaining signals compared to the WT controls, or 'compromised' with significant activity decrease close to or indistinguishable in readout from deletion controls.

Table S2 . Residues at the active center of DPH1 and DPH2
(Dong et al., 2018)alyses in yeast define cysteines that contribute to the FeS clusters(Dong et al. 2019).(B)structuralanalyses of Cmn.DPH2/2 proposes residues that are involved in SAM binding(Dong et al., 2018).